Human membrane recycling proteins

ABSTRACT

The invention provides human membrane recycling proteins (HMRP) and polynucleotides which identify and encode HMRP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HMRP.

This application is a divisional application of U.S. application Ser.No. 09/004,502, filed Jan. 8, 1998 now U.S. Pat. No. 5,962,263.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of humanmembrane recycling proteins, and to the use of these sequences in thediagnosis, treatment, and prevention of neurological, endocrine,immunological and cell proliferative disorders.

BACKGROUND OF THE INVENTION

Intercellular communication is essential for the development andsurvival of multicellular organisms. Cells communicate with one anotherthrough the secretion and uptake of protein signaling molecules. Theuptake of proteins into the cell is achieved by endocytosis, in whichthe interaction of signaling molecules with the plasma membrane surface,often via binding to specific receptors, results in the formation ofplasma membrane-derived vesicles that enclose and transport themolecules into the cytosol. The secretion of proteins from the cell isachieved by exocytosis, in which molecules inside of the cell arepackaged into membrane-bound transport vesicles derived from the transGolgi network (TGN). These vesicles fuse with the plasma membrane andexpel their contents into the surrounding extracellular space.Endocytosis and exocytosis result in the removal and addition of plasmamembrane components, and the recycling of these components is essentialto maintain the integrity, identity, and functionality of both theplasma membrane and internal membrane-bound compartments.

The endocytic and exocytic pathways converge in internal membrane-boundcompartments called endosomes that function in shuttling molecules toand from the TGN and the plasma membrane. In the endocytic pathway,vesicles from the plasma membrane fuse with the endosomal compartmentwhere the internalized signaling molecules dissociate from theirreceptors. The free receptors are then recycled back to the plasmamembrane in vesicles derived from the endosomal compartment. Likewise,in the exocytic pathway, transport vesicles derived from the TGN fusewith the plasma membrane, expel their molecular cargo, and reform. Theemptied transport vesicles then recycle back to the TGN via theendosomal compartment.

Certain differentiated cell types have highly specialized and regulatedendocytic or exocytic pathways. For example, pancreatic cells packageand store digestive enzymes in TGN-derived vesicles called secretiongranules until the cells are stimulated by hormonal signals to secretethese enzymes. Similarly, mast cells of the immune system package andstore secretion granules containing histamine molecules until stimulatedby allergic signals. Neuronal cells package and store neurotransmittersin synaptic vesicles. In response to an action potential, the synapticvesicles rapidly release their neurotransmitters into the synaptic cleftby fusing with the plasma membrane. The neurotransmitters are taken backup into the neuronal cell by endocytosis, and the neurotransmitters arerecycled into synaptic vesicles via the endosomal compartment. (Sudhof,T. C. and Jahn, R. (1991) Neuron 6:665-677; and Sudhof, T. C. et al.(1993) Cell 75:1-4.)

Biochemical and immunocytological studies in rat have shown that TGN-and endosome-derived vesicles contain characteristic integral membraneproteins called SCAMPS, secretory carrier membrane proteins. (Brand, S.H. et al. (1991) J. Biol. Chem. 266:18949-18957.) SCAMP 37, inparticular, contains structural motifs that include a potentialN-terminal metal ion-binding domain; a leucine zipper domain; two zincfinger nucleotide-binding domains; and four putative membrane-spanninghelices. SCAMPs are associated with synaptic vesicles in neuronal cells;secretion granules in endocrine cells; and plasma membrane-derivedendocytic vesicles in non-specialized fibroblast cells. (Brand, S. H.and Castle, J. D. (1993) EMBO 12:3753-3761; Laurie, S. M. et al. (1993)J. Biol. Chem. 268:19110-19117.) Because of their ubiquitous presence invesicles from diverse cellular sources, it is proposed that SCAMPs mayplay a role in a general cell surface recycling mechanism by regulatingvesicular traffic to and from the TGN and plasma membrane. This rolewould be central to intercellular communication mediated byneurotransmitters, hormones, growth factors, and other signalingmolecules involved in cell proliferation and the immune response, and inneurological or endocrine function.

The discovery of new human membrane recycling proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, treatment, andprevention of neurological, endocrine, immunological and cellproliferative disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, humanmembrane recycling proteins, referred to collectively as “HMRP” andindividually as “HMRP-1” and “HMRP-2.” In one aspect, the inventionprovides a substantially purified polypeptide, HMRP, comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

The invention further provides a substantially purified variant of HMRPhaving at least 90% sequence identity to the amino acid sequences of SEQID NO:1 or SEQ ID NO:3, or to a fragment of either of these sequences.The invention also provides an isolated and purified polynucleotideencoding the polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ IDNO:1, and a fragment of SEQ ID NO:3. The invention also includes anisolated and purified polynucleotide variant having at least 90%sequence identity to the polynucleotide encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment ofSEQ ID NO:3.

Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. The inventionfurther provides an isolated and purified polynucleotide whichhybridizes under stringent conditions to the polynucleotide encoding thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and afragment of SEQ ID NO:3, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and afragment of SEQ ID NO:3.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide having a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and afragment of SEQ ID NO:4. The invention further provides an isolated andpurified polynucleotide variant having at least 90% sequence identity tothe polynucleotide comprising a polynucleotide having a sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, afragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as anisolated and purified polynucleotide which is complementary to thepolynucleotide comprising a polynucleotide having a sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQID NO:2, and a fragment of SEQ ID NO:4. The invention also provides apolynucleotide fragment useful for designing oligonucleotides or to beused as a hybridization probe which comprises a polynucleotide having asequence selected from the group consisting of nucleotides 438-462 ofSEQ ID NO:2 and nucleotides 304-330 of SEQ ID NO:4.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.In another aspect, the expression vector is contained within a hostcell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, afragment of SEQ ID NO:1, or a fragment of SEQ ID NO:3, the methodcomprising the steps of: (a) culturing the host cell containing anexpression vector containing at least a fragment of a polynucleotidesequence encoding HMRP under conditions suitable for the expression ofthe polypeptide; and (b) recovering the polypeptide from the host cellculture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HMRP having the amino acid sequence of SEQ IDNO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, or a fragment of SEQ IDNO:3 in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ IDNO:3, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:3, as wellas a purified agonist and a purified antagonist to the polypeptide.

The invention also provides a method for treating or preventing aneurological disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HMRP.

The invention also provides a method for treating or preventing anendocrine disorder, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HMRP.

The invention also provides a method for treating or preventing animmunological disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HMRP.

The invention also provides a method for treating or preventing a cellproliferative disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist of HMRP.

The invention also provides a method for detecting a polynucleotideencoding HMRP in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide sequence encoding the polypeptide comprising SEQ ID NO:1,SEQ ID NO:3, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:3 toat least one of the nucleic acids of the biological sample, therebyforming a hybridization complex; and (b) detecting the hybridizationcomplex, wherein the presence of the hybridization complex correlateswith the presence of a polynucleotide encoding HMRP in the biologicalsample. In one aspect, the nucleic acids of the biological sample areamplified by the polymerase chain reaction prior to the hybridizingstep.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of HMRP-1.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show the amino acid sequence (SEQ IDNO:3) and nucleic acid sequence (SEQ ID NO:4) of HMRP-2.

The alignments were produced using MACDNASIS PRO software (HitachiSoftware Engineering Co. Ltd., San Bruno, Calif.).

FIGS. 3A and 3B show the amino acid sequence alignments among HMRP-1(980615; SEQ ID NO:1), HMRP-2 (412453; SEQ ID NO:3), and rat SCAMP 37(GI 487057; SEQ ID NO:5). The alignments were produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

“HMRP,” as used herein, refers to the amino acid sequences ofsubstantially purified HMRP obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to HMRP, increases or prolongs the duration of the effect of HMRP.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HMRP.

An “allele” or an “allelic sequence,” as these terms are used herein, isan alternative form of the gene encoding HMRP. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

“Altered” nucleic acid sequences encoding HMRP, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as HMRP ora polypeptide with at least one functional characteristic of HMRP.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HMRP, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HMRP. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HMRP. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of HMRP isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments”, “immunogenic fragments”, or“antigenic fragments” refer to fragments of HMRP which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of HMRP. Where “amino acidsequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp.1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to HMRP, decreases the amount or the duration of the effectof the biological or immunological activity of HMRP. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HMRP.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHMRP polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic HMRP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HMRP orfragments of HMRP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase “consensus sequence,” as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HMRP, bynorthern analysis is indicative of the presence of nucleic acidsencoding HMRP in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding HMRP.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of HMRP, of a polynucleotide sequence encoding HMRP, or ofa polynucleotide sequence complementary to a polynucleotide sequenceencoding HMRP. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (LASERGENE softwarepackage, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can createalignments between two or more sequences according to different methods,e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The Clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theClustal Method, or by other methods known in the art, such as the JotunHein Method. (See, e.g., Hein, J. (1990) Methods in Enzymology183:626-645.) Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term “hybridization complex”, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

The term “microarray,” as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term “modulate,” as it appears herein, refers to a change in theactivity of HMRP. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HMRP.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimers,”“primers,” “oligomers,” and “probes,” as these terms are commonlydefined in the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HMRP,or fragments thereof, or HMRP itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport); a tissue; a tissue print; and the like.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A “variant” of HMRP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of new human membrane recyclingproteins (HMRP), the polynucleotides encoding HMRP, and the use of thesecompositions for the diagnosis, treatment, or prevention ofneurological, endocrine, immunological and cell proliferative disorders.

Nucleic acids encoding the HMRP-1 of the present invention were firstidentified in Incyte Clone 980615 from the tongue tumor cDNA library(TONGTUT01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones3841666 (DENDNOT01), 1513267 (PANCTUT01), 154957 (THPIPLB02), 1628138(COLNPOT01), 980615 (TONGTUT01), 364646 (PROSNOT01), and 1923134(BRSTTUT01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,and 1D. HMRP-1 is 347 amino acids in length and has a potential cAMP-and cGMP-dependent protein kinase phosphorylation site at residue S₇₆;two potential casein kinase II phosphorylation sites at S₁₅ and S₇₆; apotential protein kinase C phosphorylation site at S₇₂; and a leucinezipper pattern comprising amino acids L₉₉ through L₁₂₀. As shown inFIGS. 3A and 3B, HMRP-1 has chemical and structural homology with ratSCAMP 37 (GI 487057; SEQ ID NO:5). In particular, HMRP-1 shares 49%identity with rat SCAMP 37. The leucine zipper pattern of HMRP-1 shares82% identity with that of rat SCAMP 37. The region of HMRP-1 from C₁₈₅to C₂₁₆ shares 72% identity with one of the putative zinc-fingernucleotide-binding domains of rat SCAMP 37 and shows conservation of allfour potential zinc-coordinating cysteines at C₁₈₅, C₁₉₀, C₂₁₂, andC₂₁₆. In addition, hydrophobic residues within the four putativetransmembrane domains (TM) are conserved between the two proteins.Within HMRP-1, TM1 extends from about T₁₆₆ to about V₁₉₁; TM2, fromabout F₁₉₉ to about W₂₁₇; TM3, from about F₂₃₃ to about I₂₅₄; and TM4,from about T₂₇₃ to about L₂₉₇. A fragment of the HMRP-1 nucleic acidsequence useful for designing oligonucleotides or to be used directly asa hybridization probe to distinguish between the sequences encodingHMRP-1 and rat SCAMP 37 comprises nucleotides 438-462. Northern analysisshows the expression of HMRP-1 in various libraries, at least 64% ofwhich involve cell proliferation and at least 27% of which involveimmune response. Of particular note is the expression of HMRP-1 inreproductive, neuronal, gastrointestinal, and hematopoietic tissues thatactively employ transport vesicle-mediated secretion mechanisms.

Nucleic acids encoding the HMRP-2 of the present invention were firstidentified in Incyte Clone 412453 from the breast tissue cDNA library(BRSTNOT01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:4, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones1256847 (MENITUT03), 639585 (BRSTNOT03), 1333295 (COLNNOT13), 1435380(PANCNOT08), 412453 (BRSTNOT01), 2873157 (THYRNOT10), 776678(COLNNOT05), 834309 (PROSNOT07), 815923 (OVARTUT01), and 1240683(LUNGNOT03).

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2A,2B, 2C, 2D, 2E, and 2F. HMRP-2 is 329 amino acids in length and has twopotential N-glycosylation sites at N₁₇₇ and N₂₅₆; two potential caseinkinase II phosphorylation sites at S₂ and S₂₅₂; two potential proteinkinase C phosphorylation sites at S₁₇₈ and S₃₁₀; and two leucine zipperpatterns comprising amino acids L₈₅ through L₁₀₆ and L₉₂ through L₁₁₃.As shown in FIGS. 3A and 3B, HMRP-2 has chemical and structural homologywith rat SCAMP 37 (GI 487057; SEQ ID NO:5). In particular, HMRP-2 shares50% identity with rat SCAMP 37. The leucine zipper pattern of HMRP-2shares 73% identity with that of rat SCAMP 37. The region of HMRP-2 fromC₁₇₀ to C₂₀₁, shares 75% identity with one of the putative zinc-fingernucleotide-binding domains of rat SCAMP 37 and shows conservation ofthree of the four potential zinc-coordinating cysteines at C₁₇₀, C₁₉₇,and C₂₀₁. Residues D₅, N₇, F₉, and D₁₁ of HMRP-2 are identical to fourof six putative metal ion-coordinating residues in the equivalentpositions of the rat SCAMP 37 N-terminus. The potential phosphorylationsites at S₂, S178, and S₃₁₀ are conserved between the two proteins. Inaddition, hydrophobic residues within the four putative transmembranedomains (TM) are conserved. Within HMRP-2, TM1 extends from about M₁₅₁to about G₁₇₆; TM2, from about F₁₈₄ to about W₂₀₂; TM3, from about F₂₁₈to about I₂₃₉; and TM4, from about L₂₅₉ to about L₂₈₃. A fragment of theHMRP-2 nucleic acid sequence useful for designing oligonucleotides or tobe used directly as a hybridization probe to distinguish between thesequences encoding HMRP-2 and rat SCAMP 37 comprises nucleotides304-330. Northern analysis shows the expression of HMRP-2 in variouslibraries, at least 66% of which involve cell proliferation and at least32% of which involve immune response. Of particular note is theexpression of HMRP-2 in reproductive, gastrointestinal, hematopoietic,and neuronal tissues that actively employ transport vesicle-mediatedsecretion mechanisms.

The invention also encompasses HMRP variants. A preferred HMRP variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HMRP amino acid sequence, and which contains at least one functionalor structural characteristic of HMRP.

The invention also encompasses polynucleotides which encode HMRP. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes anHMRP-1, as shown in FIGS. 1A, 1B, 1C, and 1D. In a further embodiment,the invention encompasses a polynucleotide sequence comprising thesequence of SEQ ID NO:4, which encodes an HMRP-2, as shown in FIGS. 2A,2B, 2C, 2D, 2E, and 2F.

The invention also encompasses a variant of a polynucleotide sequenceencoding HMRP. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HMRP. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. The inventionfurther encompasses a polynucleotide variant of SEQ ID NO:4 having atleast about 80%, more preferably at least about 90%, and most preferablyat least about 95% polynucleotide sequence identity to SEQ ID NO:4. Anyone of the polynucleotide variants described above can encode an aminoacid sequence which contains at least one functional or structuralcharacteristic of HMRP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HMRP, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HMRP, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HMRP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HMRP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HMRP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HMRP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeHMRP and HMRP derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HMRP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, SEQ ID NO:4, a fragment ofSEQ ID NO:2, or a fragment of SEQ ID NO:4, under various conditions ofstringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) MethodsEnzymol. 152:399-407; and Kimmel, A. R. (1987) Methods Enzymol.152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO/BRL Gaithersburg, Md.). Preferably, the process is automated withmachines such as the MICROLAB 2200 (Hamilton, Reno, Nev.), Peltierthermal cycler (PTC200; MJ Research, Watertown, Mass.), and the ABICatalyst and 373 and 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding HMRP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer to a linker sequence and a primerspecific to the known region. The amplified sequences are then subjectedto a second round of PCR with the same linker primer and anotherspecific primer internal to the first one. Products of each round of PCRare transcribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etal. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR, nested primers, PROMOTERFINDER libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This process avoids the need to screenlibraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HMRP may be used in recombinant DNAmolecules to direct expression of HMRP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHMRP.

As will be understood by those of skill in the art, it may beadvantageous to produce HMRP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HMRP encodingsequences for a variety of reasons including, but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HMRP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HMRP activity, it may be useful toencode a chimeric HMRP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HMRP encoding sequence and theheterologous protein sequence, so that HMRP may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HMRP may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223; and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of HMRP, or afragment thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques. (Roberge, J. Y. et al. (1995) Science269:202-204.) Automated synthesis may be achieved using the ABI 431 Apeptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., Creighton, T. (1983) Proteins,Structures and Molecular Properties, WH Freeman and Co., New York, N.Y.)Additionally, the amino acid sequence of HMRP, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active HMRP, the nucleotide sequencesencoding HMRP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HMRP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HMRP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector (i.e., enhancers, promoters, and 5′and 3′ untranslated regions) which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO, and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HMRP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HMRP. For example, when largequantities of HMRP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HMRP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (See, e.g., Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509.); pGEX vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-544.)

In cases where plant expression vectors are used, the expression ofsequences encoding HMRP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al.(1991) Results Probl. Cell Differ. 17:85-105.) These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews. (See, e.g., Hobbs, S. or Murry,L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGrawHill, New York, N.Y.; pp. 191-196.)

An insect system may also be used to express HMRP. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HMRP may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of HMRP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which HMRP may be expressed. (See, e.g., Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HMRP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HMRP in infected host cells. (See, e.g., Logan, J.and T. Shenk (1984) Proc. Nati. Acad. Sci. 81:3655-3659.) In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HMRP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HMRP and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing HMRP can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes and adenine phosphoribosyltransferase genes,which can be employed in tk⁻ or apr⁻ cells, respectively. (See, e.g.,Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980)Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate; npt confers resistance to theaminoglycosides neomycin and G-418; and als or pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively. (See,e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570;Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry,supra.) Additional selectable genes have been described, e.g., trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. (See,e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.85:8047-8051.) Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, β glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin. These markerscan be used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHMRP is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HMRP can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HMRP under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HMRP and express HMRP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding HMRP can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HMRP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HMRP to detect transformantscontaining DNA or RNA encoding HMRP.

A variety of protocols for detecting and measuring the expression ofHMRP, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HMRP is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HMRP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HMRP,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Micg.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HMRP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHMRP may be designed to contain signal sequences which direct secretionof HMRP through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HMRP to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the HMRP encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HMRP and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography. (IMIAC) (See, e.g., Porath, J. et al. (1992)Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage site provides ameans for purifying HMRP from the fusion protein. (See, e.g., Kroll, D.J. et al. (1993) DNA Cell Biol. 12:441-453.)

Fragments of HMRP may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Proteinsynthesis may be performed by manual techniques or by automation.Automated synthesis may be achieved, for example, using the AppliedBiosystems 431 A peptide synthesizer (Perkin Elmer). Various fragmentsof HMRP may be synthesized separately and then combined to produce thefull length molecule.

Therapeutics

Chemical and structural homology exists between HMRP and rat SCAMP 37(GI 487057). In addition, HMRP is widely expressed in proliferatingtissues and in tissues associated with the immune system. Therefore,HMRP appears to play a role in neurological, endocrine, immunologicaland cell proliferative disorders.

Therefore, in one embodiment, HMRP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a neurologicaldisorder. Such disorders can include, but are not limited to, akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolardisorder, catatonia, cerebral neoplasms, dementia, depression, diabeticneuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson'sdisease, paranoid psychoses, postherpetic neuralgia, schizophrenia, andTourette's disorder.

In another embodiment, a vector capable of expressing HMRP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a neurological disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HMRP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a neurological disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHMRP may be administered to a subject to treat or prevent a neurologicaldisorder including, but not limited to, those listed above.

In another embodiment, HMRP or a fragment or derivative thereof may beadministered to a subject to treat or prevent an endocrine disorder.Such disorders can include, but are not limited to, Addison's disease,carcinoid syndrome, Cushing's disease, diabetes insipidus, diabetesmellitus, hyperaldosteronism, hyper- and hypoglycemia, goiter, Grave'sdisease, multiple endocrine neoplasia syndromes, pheochromocytoma,polyendocrine deficiency syndromes, and thryoiditis.

In another embodiment, a vector capable of expressing HMRP or a fragmentor derivative thereof may be administered to a subject to treat orprevent an endocrine disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HMRP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an endocrine disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHMRP may be administered to a subject to treat or prevent an endocrinedisorder including, but not limited to, those listed above.

In another embodiment, HMRP or a fragment or derivative thereof may beadministered to a subject to treat or prevent an immunological disorder.Such disorders can include, but are not limited to, AIDS, Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermayitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome,systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,ulcerative colitis, Werner syndrome, and complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma.

In another embodiment, a vector capable of expressing HMRP or a fragmentor derivative thereof may be administered to a subject to treat orprevent an immunological disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HMRP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an immunological disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHMRP may be administered to a subject to treat or prevent animmunological disorder including, but not limited to, those listedabove.

In a further embodiment, an antagonist of HMRP may be administered to asubject to treat or prevent a cell proliferative disorder. Suchdisorders may include, but are not limited to, arteriosclerosis,atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissuedisease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,polycythemia vera, psoriasis, primary thrombocythemia, and cancersincluding adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds HMRP may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HMRP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HMRP may be administered to a subject to treator prevent a cell proliferative disorder including, but not limited to,those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HMRP may be produced using methods which are generallyknown in the art. In particular, purified HMRP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HMRP. Antibodies to HMRP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HMRP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HMRP have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HMRP amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to HMRP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HMRP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:11120-11123.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HMRP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 254:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HMRP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HMRP epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingHMRP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHMRP may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HMRP. Thus,complementary molecules or fragments may be used to modulate HMRPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding HMRP.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencecomplementary to the polynucleotides of the gene encoding HMRP. (See,e.g., Sambrook, supra; and Ausubel, supra.)

Genes encoding HMRP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof encoding HMRP. Such constructs may be used to introduceuntranslatable sense or antisense sequences into a cell. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHMRP. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules specifically andefficiently catalyze endonucleolytic cleavage of sequences encodingHMRP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HMRP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HMRP,antibodies to HMRP, and mimetics, agonists, antagonists, or inhibitorsof HMRP. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HMRP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays of neoplastic cells, forexample, or in animal models, usually mice, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HMRP or fragments thereof, antibodies of HMRP,and agonists, antagonists or inhibitors of HMRP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of topic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD50/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 μg to 100,000 μg, up to a totaldose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HMRP may beused for the diagnosis of disorders characterized by expression of HMRP,or in assays to monitor patients being treated with HMRP or agonists,antagonists, and inhibitors of HMRP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as those described above fortherapeutics. Diagnostic assays for HMRP include methods which utilizethe antibody and a label to detect HMRP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent joiningwith a reporter molecule. A wide variety of reporter molecules, severalof which are described above, are known in the art and may be used.

A variety of protocols for measuring HMRP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HMRP expression. Normal or standard values for HMRPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHMRP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods,preferably by photometric means. Quantities of HMRP expressed in subjectsamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHMRP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHMRP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HMRP,and to monitor regulation of HMRP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HMRP or closely related molecules may be used to identifynucleic acid sequences which encode HMRP. The specificity of the probe,whether it is made from a highly specific region (e.g., the 5′regulatory region) or from a less specific region (e.g., the 3′ codingregion), and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HMRP, alleles, orrelated sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HMRP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequences of SEQID NO:2 or SEQ ID NO:4 or from genomic sequences including promoter andenhancer elements and introns of the naturally occurring HMRP.

Means for producing specific hybridization probes for DNAs encoding HMRPinclude the cloning of polynucleotide sequences encoding HMRP or HMRPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HMRP may be used for the diagnosis ofa disorder associated with expression of HMRP. Examples of such adisorder include, but are not limited to, a neurological disorder suchas akathesia, Alzheimer's disease, amnesia, arnyotrophic lateralsclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia,depression, diabetic neuropathy, Down's syndrome, tardive dyskinesia,dystonias, epilepsy, Huntington's disease, multiple sclerosis,neurofibromatosis, Parkinson's disease, paranoid psychoses, postherpeticneuralgia, schizophrenia, and Tourette's disorder; an endocrine disordersuch as Addison's disease, carcinoid syndrome, Cushing's disease,diabetes insipidus, diabetes mellitus, hyperaldosteronism, hyper- andhypoglycemia, goiter, Grave's disease, multiple endocrine neoplasiasyndromes, pheochromocytoma, polyendocrine deficiency syndromes, andthryoiditis; an immunological disorder such as AIDS, Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermayitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome,systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,ulcerative colitis, Werner syndrome, and complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma; and a cellproliferative disorder such as arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding HMRP may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patient biopsies to detect altered HMRP expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HMRP may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHMRP may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered from that of a comparable control sample, the nucleotidesequences have hybridized with nucleotide sequences in the sample, andthe presence of altered levels of nucleotide sequences encoding HMRP inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orin monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HMRP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HMRP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for a disorder. Deviation from standard values isused to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HMRP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HMRP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HMRP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HMRPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously (to produce atranscript image) and to identify genetic variants, mutations, andpolymorphisms. This information may be used in determining genefunction, in understanding the genetic basis of a disorder, indiagnosing a disorder, and in developing and monitoring the activitiesof therapeutic agents.

In one embodiment, the microarray is prepared and used according tomethods known in the art. (See, e.g., Chee et al. (1995) PCT applicationW095/11995; Lockhart, D. J. et al. (1996) Nat. Biotech. 14:1675-1680;and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619.)

The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6 to 60 nucleotidesin length, more preferably about 15 to 30 nucleotides in length, andmost preferably about 20 to 25 nucleotides in length. For a certain typeof microarray, it may be preferable to use oligonucleotides which areabout 7 to 10 nucleotides in length. The microarray may containoligonucleotides which cover the known 5′ or 3′ sequence, or may containsequential oligonucleotides which cover the full length sequence orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest in which atleast a fragment of the sequence is known or oligonucleotides specificto one or more unidentified cDNAs common to a particular cell or tissuetype or to a normal, developmental, or disease state. In certainsituations, it may be appropriate to use pairs of oligonucleotides on amicroarray. The pairs will be identical, except for one nucleotidepreferably located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from about 2 to 1,000,000.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ end, or, more preferably, at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon, any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

In one aspect, the oligonucleotides may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus. (See, e.g., Baldeschweiler et al. (1995) PCTapplication WO95/251116.) In another aspect, a grid array analogous to adot or slot blot (HYBRIDOT apparatus, GIBCO/BRL) may be used to arrangeand link cDNA fragments or oligonucleotides to the surface of asubstrate using a vacuum system or thermal, UV, mechanical or chemicalbonding procedures. In yet another aspect, an array may be produced byhand or by using available devices, materials, and machines (includingBRINKMAN multichannel pipettors or robotic instruments), and may contain8, 24, 96, 384, 1536, or 6144 oligonucleotides, or any other multiplefrom 2 to 1,000,000 which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce nucleic acid sequences which arecomplementary to the nucleic acids on the microarray. If the microarrayconsists of cDNAs, antisense RNAs (aRNA) are appropriate probes.Therefore, in one aspect, mRNA is used to produce cDNA which, in turnand in the presence of fluorescent nucleotides, is used to producefragment or oligonucleotide aRNA probes. These fluorescently labeledprobes are incubated with the microarray so that the probe sequenceshybridize to the cDNA oligonucleotides of the microarray. In anotheraspect, nucleic acid sequences used as probes can includepolynucleotides, fragments, and complementary or antisense sequencesproduced using restriction enzymes, PCR technologies, and Oligolabelingor TransProbe kits (Pharmacia & Upjohn) well known in the area ofhybridization technology.

Incubation conditions are adjusted so that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity. After removal of nonhybridized probes, a scanner isused to determine the levels and patterns of fluorescence. The scannedimages are examined to determine the degree of complementarity and therelative abundance of each oligonucleotide sequence on the microarray. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or for functional analysis of the sequences, mutations,variants, or polymorphisms among samples. (See, e.g., Heller, R. A. etal. (1997) Proc. Natl. Acad. Sci. 94:2150-2155.)

In another embodiment of the invention, nucleic acid sequences encodingHMRP may be used to generate hybridization probes useful for mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, such as human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev.7:127-134; and Trask, B. J. (1991) Trends Genet.

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HMRP on a physical chromosomal map and aspecific disorder, or predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the subject invention may be used to detect differences ingene sequences between normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., AT to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, HMRP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HMRPand the agent being tested may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest. (See, e.g., Geysen, et al. (1984) PCTapplication WO84/03564.) In this method, large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withHMRP, or fragments thereof, and washed. Bound HMRP is then detected bymethods well known in the art. Purified HMRP can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HMRP specificallycompete with a test compound for binding HMRP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HMRP.

In additional embodiments, the nucleotide sequences which encode HMRPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. cDNA Library Construction

TONGTUT01

The TONGTUT01 cDNA library was constructed from tongue tumor tissueremoved from a 36-year-old Caucasian male. The frozen tissue washomogenized and lysed in guanidinium isothiocyanate solution using ahomogenizer Polytron-PT 3000 (Brinkmann Instruments Inc, Westbury,N.Y.). The lysate was extracted once with phenol, and RNA was isolatedaccording to Stratagene's RNA isolation protocol (Stratagene, La Jolla,Calif.). The RNA was extracted twice with phenol, precipitated withsodium acetate and ethanol, resuspended in RNase-free water, and DNasetreated. Poly(A+) RNA was isolated using the OLIGOTX kit (QIAGEN Inc,Chatsworth, Calif.).

Poly(A+) RNA was used to construct the TONGTUT01 cDNA library accordingto the recommended protocols in the SUPERSCRIPT plasmid system for cDNAsynthesis and plasmid cloning (Cat. #18248-013, Gibco/BRL, Gaithersburg,Md.). The cDNAs were fractionated on a SEPHAROSE CL4B column (Cat.#275105-01, Pharmacia, Piscataway, N.J.), and those cDNAs exceeding 400bp were ligated into the PSPORT1 plasmid (Gibco/BRL). PSPORT1 wassubsequently transformed into DH5α competent cells (Cat. #18258-012,Gibco/BRL).

BRSTNOT01

The BRSTNOT01 cDNA library was constructed from breast tissue removedfrom a 56-year-old Caucasian female who died an accidental death (lot#93032903, Keystone Skin Bank, International Institute for theAdvancement of Medicine, Exton, Pa.). The frozen tissue was ground witha mortar and pestle and lysed in guanidinium isothiocyanate solution.The lysate was extracted twice with phenol/chloroform and centrifugedover a CsCl cushion to isolate RNA. The RNA was precipitated with sodiumacetate and ethanol, resuspended in RNase-free water, and DNase treated.Poly(A+) RNA was isolated using the OLIGOTEX kit (QIAGEN Inc)

Poly(A+) RNA was used to construct the BRSTNOT01 cDNA library. Firststrand cDNA synthesis was achieved using an oligo-d(T) primer containingan XhoI restriction site. Second strand synthesis was achieved using acombination of DNA polymerase I, E. coli ligase, and RNase H, followedby the ligation of an EcoRI adaptor to the blunt-ended cDNA. The cDNAwas then digested with XhoI restriction enzyme and fractionated onSEPHACRYL S400 (Pharmacia) to obtain sequences that exceeded 1000 bp insize. The size selected cDNAs were inserted into the LAMBDAZAP vectorsystem (Stratagene) and transformed into E. coli strain XL1-BLUEMRF™(Stratagene).

The PBLUESCRIPT phagemid forms of individual cDNA clones were obtainedby the in vivo excision process (Stratagene). The phagemid DNA waspurified and used to reinfect fresh host cells (SOLR, Stratagene) toallow recovery of plasmid DNA

II Isolation and Sequencing of cDNA Clones

TONGTUT01

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid kit (Cat. #26173, QIAGEN Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: I) the bacteria were cultured in 1 mlof sterile Terrific Broth (Cat. #22711, Gibco/BRL) with carbenicillin at25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and then lysed with 0.3 ml of lysis buffer; and3) following isopropanol precipitation, the plasmid DNA pellets wereeach resuspended in 0.1 ml of distilled water. The plasmid DNA sampleswere stored at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with peltier thermal cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA sequencing systems.

BRSTNOT01

Plasmid DNA was released from the cells and purified using the MINIPREPkit (Cat. #77468; Advanced Genetic Technologies Corporation,Gaithersburg, Md.). This kit enabled the simultaneous purification of 96samples in a 96-well block using multi-channel reagent dispensers. Therecommended protocol was employed except for the following changes: 1)the bacteria were cultured in 1 ml of sterile Terrific Broth (Cat.#22711, Gibco/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%;2) after inoculation, the cultures were incubated for 24 hours and thenlysed with 60 μl of lysis buffer; 3) the contents of the block werecentrifuged in the Beckman GS-6R (2900 rpm, 5 minutes) before theiraddition to the primary filter plate; and 4) the addition of isopropanolto TRIS buffer was not routinely performed. After the last step in theprotocol, plasmid DNA samples were stored at 4° C.

The cDNAs were sequenced as were the TONGTUT01 cDNAs described above.

III. Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁰ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam).Deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, F. M. et al. supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor the LIFESEQ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:

% sequence identity×% maximum BLAST score 100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact.Homologous molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HMRP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HMRP Encoding Polynucleotides

The nucleic acid sequences of Incyte Clones 980615 and 412453 were usedto design oligonucleotide primers for extending partial nucleotidesequences to full length. For each nucleic acid sequence, one primer wassynthesized to initiate extension of an antisense polynucleotide strand,and the other was synthesized to initiate extension of a sensepolynucleotide strand. Primers were used to facilitate the extension ofthe known sequence “outward” generating amplicons containing new unknownnucleotide sequence for the region of interest. The initial primers weredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be about 22 to 30 nucleotides inlength, to have a GC content of about 50% or more, and to anneal to thetarget sequence at temperatures of about 68° C. to about 72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier thermal cycler(PTC200; M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (andholding)

A 5 l to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN), and trimmed of overhangsusing Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing 2×Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2×Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:4are used to obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer and 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN,Boston, Mass.). The labeled oligonucleotides are substantially purifiedusing a SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn). Analiquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUSE, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, or the bots areexposed in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale,Calif.) hybridization patterns are compared visually.

VII. Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3′ end of the nucleotide sequence. Thealgorithm identifies oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies approximately 20sequence-specific oligonucleotides of 20 nucleotides in length(20-mers). A matched set of oligonucleotides are created in which onenucleotide in the center of each sequence is altered. This process isrepeated for each gene in the microarray, and double sets of twenty20-mers are synthesized and arranged on the surface of the silicon chipusing a light-directed chemical process. (See, e.g., Chee, supra.)

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate. (See,e.g., Baldeschweiler, supra.) In another alternative, a grid arrayanalogous to a dot or slot blot is used to arrange and link cDNAfragments or oligonucleotides to the surface of a substrate using avacuum system or thermal, UV, mechanical, or chemical bondingprocedures. A typical array may be produced by hand or using availablematerials and machines and contain grids of 8 dots, 24 dots, 96 dots,384 dots, 1536 dots, or 6144 dots. After hybridization, the microarrayis washed to remove nonhybridized probes, and a scanner is used todetermine the levels and patterns of fluorescence. The scanned image isexamined to determine the degree of complementarity and the relativeabundance/expression level of each oligonucleotide sequence in themicroarray.

VIII. Complementary Polynucleotides

Sequences complementary to the HMRP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HMRP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using Oligo 4.06 software andthe coding sequence of HMRP. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HMRP-encoding transcript.

IX. Expression of HMRP

Expression of HMRP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express HMRP in E. coli.This vector contains a promoter for β-galactosidase upstream of thecloning site, followed by sequence containing the amino-terminal Met andthe subsequent seven residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HMRP into bacterialgrowth media which can be used directly in the following assay foractivity.

X. Demonstration of HMRP Activity

HMRP activity is measured by the ability of epitope-tagged HMRP tolocalize to recycling endosomes in African green monkey kidney (Vero)cells. (Wu, T. T. and Castle, J. D. (1997) J. Cell Sci. 110:1533-1541.)In this assay, a DNA sequence encoding the myc epitope tag is insertedin frame into the HMRP cDNA immediately preceding the N-terminalmethionine codon using recombinant DNA methods well known in the art.This construct is transfected into Vero cells using an appropriateeukaryotic expression vector. Expressed epitope-tagged HMRP is detectedin situ by immunofluorescence microscopy using antibody directed againstthe myc epitope. The localization of epitope-tagged HMRP reflects thatof endogenous HMRP, as previous studies have shown that N-terminal mycepitope-tagged SCAMP 37 colocalizes with endogenous SCAMP-37. (Wu,supra.) Antibody H68.4 directed against human transferrin receptor isused to label recycling endosomes in transfected cells. Co-localizationof myc-tagged HMRP and transferrin receptor iz demonstrated by confocallaser scanning microscopy and imaging analysis and is indicative oflocalization of myc-tagged HMRP to recycling endosomes.

XI. Production of HMRP Specific Antibodies

HMRP substantially purified using PAGE electrophoresis (see, e.g.,Harrington, M. G. (1990) Methods Enzymol. 182:488495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The HMRP amino acid sequence isanalyzed using DNASTAR software (DNASTAR, Inc.) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). (See, e.g.,Ausubel et al. supra.) Rabbits are immunized with the oligopeptide-KLHcomplex in complete Freund's adjuvant. Resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio-iodinated goat anti-rabbit IgG.

XII. Purification of Naturally Occurring HMRP Using Specific Antibodies

Naturally occurring or recombinant HMRP is substantially purified byimmunoaffinity chromatography using antibodies specific for HMRP. Animmunoaffinity column is constructed by covalently coupling HMRPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HMRP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HMRP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HMRP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HMRPis collected.

XIII. Identification of Molecules Which Interact with HMRP

HMRP or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HMRP, washed, and anywells with labeled HMRP complex are assayed. Data obtained usingdifferent concentrations of HMRP are used to calculate values for thenumber, affinity, and association of HMRP with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

5 347 amino acids amino acid single linear unknown TONGTUT01 980615 1Met Ala Gln Ser Arg Asp Gly Gly Asn Pro Phe Ala Glu Pro Ser Glu 1 5 1015 Leu Asp Asn Pro Phe Gln Asp Pro Ala Val Ile Gln His Arg Pro Ser 20 2530 Arg Gln Tyr Ala Thr Leu Asp Val Tyr Asn Pro Phe Glu Thr Arg Glu 35 4045 Pro Pro Pro Ala Tyr Glu Pro Pro Ala Pro Ala Pro Leu Pro Pro Pro 50 5560 Ser Ala Pro Ser Leu Gln Pro Ser Arg Lys Leu Ser Pro Thr Glu Pro 65 7075 80 Lys Asn Tyr Gly Ser Tyr Ser Thr Gln Ala Ser Ala Ala Ala Ala Thr 8590 95 Ala Glu Leu Leu Lys Lys Gln Glu Glu Leu Asn Arg Lys Ala Glu Glu100 105 110 Leu Asp Arg Arg Glu Arg Glu Leu Gln His Ala Ala Leu Gly GlyThr 115 120 125 Ala Thr Arg Gln Asn Asn Trp Pro Pro Leu Pro Ser Phe CysPro Val 130 135 140 Gln Pro Cys Phe Phe Gln Asp Ile Ser Met Glu Ile ProGln Glu Phe 145 150 155 160 Gln Lys Thr Val Ser Thr Met Tyr Tyr Leu TrpMet Cys Ser Thr Leu 165 170 175 Ala Leu Leu Leu Asn Phe Leu Ala Cys LeuAla Ser Phe Cys Val Glu 180 185 190 Thr Asn Asn Gly Ala Gly Phe Gly LeuSer Ile Leu Trp Val Leu Leu 195 200 205 Phe Thr Pro Cys Ser Phe Val CysTrp Tyr Arg Pro Met Tyr Lys Ala 210 215 220 Phe Arg Ser Asp Ser Ser PheAsn Phe Phe Val Phe Phe Phe Ile Phe 225 230 235 240 Phe Val Gln Asp ValLeu Phe Val Leu Gln Ala Ile Gly Ile Pro Gly 245 250 255 Trp Gly Phe SerGly Trp Ile Ser Ala Leu Val Val Pro Lys Gly Asn 260 265 270 Thr Ala ValSer Val Leu Met Leu Leu Val Ala Leu Leu Phe Thr Gly 275 280 285 Ile AlaVal Leu Gly Ile Val Met Leu Lys Arg Ile His Ser Leu Tyr 290 295 300 ArgArg Thr Gly Ala Ser Phe Gln Lys Ala Gln Gln Glu Phe Ala Ala 305 310 315320 Gly Val Phe Ser Asn Pro Ala Val Arg Thr Ala Ala Ala Asn Ala Ala 325330 335 Ala Gly Ala Ala Glu Asn Ala Phe Arg Ala Pro 340 345 1521 basepairs nucleic acid single linear unknown TONGTUT01 980615 2 NGACGCAGGCGCAACCCACG GCTGCTGCGG GGATCCTTGT GGCCCTTCCG GTCGATGGAA 60 CCAATCCGTGCACAGAGAAG CGGGGCGAAC TGAGGCGAGT GAAGTGGACT CTGAGGGCTA 120 CCGCTACCGCCACTGCTGCG GCAGGGGCGT GGAGGGCAGA GGGCCGCGGA GGCCGCAGTT 180 GCAAACATGGCTCAGAGCAG AGACGGCGGA AACCCGTTCG CCGAGCCCAG CGAGCTTGAC 240 AACCCCTTTCAGGACCCAGC TGTGATCCAG CACCGACCCA GCCGGCAGTA TGCCACGCTT 300 GACGTCTACAACCCTTTTGA GACCCGGGAG CCACCACCAG CCTATGAGCC TCCAGCCCCT 360 GCCCCATTGCCTCCACCCTC AGCTCCCTCC TTGCAGCCCT CGAGAAAGCT CAGCCCCACA 420 GAACCTAAGAACTATGGCTC ATACAGCACT CAGGCCTCAG CTGCAGCAGC CACAGCTGAG 480 CTGCTGAAGAAACAGGAGGA GCTCAACCGG AAGGCAGAGG AGTTGGACCG AAGGGAGCGA 540 GAGCTGCAGCATGCTGCCCT GGGGGGCACA GCTACTCGAC AGAACAATTG GCCCCCTCTA 600 CCTTCTTTTTGTCCAGTTCA GCCCTGCTTT TTCCAGGACA TCTCCATGGA GATCCCCCAA 660 GAATTTCAGAAGACTGTATC CACCATGTAC TACCTCTGGA TGTGCAGCAC GCTGGCTCTT 720 CTCCTGAACTTCCTCGCCTG CCTGGCCAGC TTCTGTGTGG AAACCAACAA TGGCGCAGGC 780 TTTGGGCTTTCTATCCTCTG GGTCCTCCTT TTCACTCCCT GCTCCTTTGT CTGCTGGTAC 840 CGCCCCATGTATAAGGCTTT CCGGAGTGAC AGTTCATTCA ATTTCTTCGT TTTCTTCTTC 900 ATTTTCTTCGTCCAGGATGT GCTCTTTGTC CTCCAGGCCA TTGGTATCCC AGGTTGGGGA 960 TTCAGTGGCTGGATCTCTGC TCTGGTGGTG CCGAAGGGCA ACACAGCAGT ATCCGTGCTC 1020 ATGCTGCTGGTCGCCCTGCT CTTCACTGGC ATTGCTGTGC TAGGAATTGT CATGCTGAAA 1080 CGGATCCACTCCTTATACCG CCGCACAGGT GCCAGCTTTC AGAAGGCCCA GCAAGAATTT 1140 GCTGCTGGTGTCTTCTCCAA CCCTGCGGTG CGAACCGCAG CTGCCAATGC AGCCGCTGGG 1200 GCTGCTGAAAATGCCTTCCG GGCCCCGTGA CCCCTGACTG GGATGCCCTG GCCCTGCTAC 1260 TTGAGGGAGCTGACTTAGCT CCCGTCCCTA AGGTCTCTGG GACTTGGAGA GACATCACTA 1320 ACTGATGGCTCCTCCGTAGT GCTCCCAATC CTATGGCCAT GACTGCTGAA CCTGACAGGC 1380 GTGTGGGGAGTTCACTGTGA CCTAGTCCCC CCATCAGGCC ACACTGCTGC CACCTCTCAC 1440 ACGCCCCAACCCAGCTTCCC TCTGCTGTGC CACGGCTGTT GCTTCGGTTA TTTAAATAAA 1500 AAGAAAGTGGAACTGGAACT G 1521 329 amino acids amino acid single linear unknownBRSTNOT01 412453 3 Met Ser Ala Phe Asp Thr Asn Pro Phe Ala Asp Pro ValAsp Val Asn 1 5 10 15 Pro Phe Gln Asp Pro Ser Val Thr Gln Leu Thr AsnAla Pro Gln Gly 20 25 30 Gly Leu Ala Glu Phe Asn Pro Phe Ser Glu Thr AsnAla Ala Thr Thr 35 40 45 Val Pro Val Thr Gln Leu Pro Gly Ser Ser Gln ProAla Val Leu Gln 50 55 60 Pro Ser Val Glu Pro Thr Gln Pro Thr Pro Gln AlaVal Val Ser Ala 65 70 75 80 Ala Gln Ala Gly Leu Leu Arg Gln Gln Glu GluLeu Asp Arg Lys Ala 85 90 95 Ala Glu Leu Glu Arg Lys Glu Arg Glu Leu GlnAsn Thr Val Ala Asn 100 105 110 Leu His Val Arg Gln Asn Asn Trp Pro ProLeu Pro Ser Trp Cys Pro 115 120 125 Val Lys Pro Cys Phe Tyr Gln Asp PheSer Thr Glu Ile Pro Ala Asp 130 135 140 Tyr Gln Arg Ile Cys Lys Met LeuTyr Tyr Leu Trp Met Leu His Ser 145 150 155 160 Val Thr Leu Phe Leu AsnLeu Leu Ala Cys Leu Ala Trp Phe Ser Gly 165 170 175 Asn Ser Ser Lys GlyVal Asp Phe Gly Leu Ser Ile Leu Trp Phe Leu 180 185 190 Ile Phe Thr ProCys Ala Phe Leu Cys Trp Tyr Arg Pro Ile Tyr Lys 195 200 205 Ala Phe ArgSer Asp Asn Ser Phe Ser Phe Phe Val Phe Phe Phe Val 210 215 220 Phe PheCys Gln Ile Gly Ile Tyr Ile Ile Gln Leu Val Gly Ile Pro 225 230 235 240Gly Leu Gly Asp Ser Gly Trp Ile Ala Ala Leu Ser Thr Leu Asp Asn 245 250255 His Ser Leu Ala Ile Ser Val Ile Met Met Val Val Ala Gly Phe Phe 260265 270 Thr Leu Cys Ala Val Leu Ser Val Phe Leu Leu Gln Arg Val His Ser275 280 285 Leu Tyr Arg Arg Thr Gly Ala Ser Phe Gln Gln Ala Gln Glu GluPhe 290 295 300 Ser Gln Gly Ile Phe Ser Ser Arg Thr Phe His Arg Ala AlaSer Ser 305 310 315 320 Ala Ala Gln Gly Ala Phe Gln Gly Asn 325 2434base pairs nucleic acid single linear unknown BRSTNOT01 412453 4NCCGGAAGTG GAGGGTCTAC ACGAAGCGCC GCTGGGTCTG GGTGCCCGGA GGCAGCAGCG 60TTCGCGGAGT TCGCCCGCTG GCCCCCGATC ACCATGTCGG CTTTCGACAC CAACCCCTTC 120GCGGACCCAG TGGATGTAAA CCCCTTCCAG GATCCCTCTG TGACCCAGCT GACCAACGCC 180CCGCAGGGCG GCCTGGCGGA ATTCAACCCC TTCTCAGAGA CAAATGCAGC GACAACAGTT 240CCTGTCACCC AACTCCCTGG GTCCTCACAG CCAGCGGTTC TCCAGCCATC AGTGGAACCA 300ACCCAGCCGA CCCCCCAGGC CGTGGTGTCT GCAGCCCAGG CAGGCCTGCT CCGGCAGCAG 360GAAGAACTGG ACAGGAAAGC TGCCGAGCTG GAACGCAAGG AGCGGGAGCT GCAGAACACT 420GTAGCCAACT TGCATGTGAG ACAGAACAAC TGGCCCCCTC TGCCCTCGTG GTGCCCTGTG 480AAGCCCTGCT TCTATCAGGA TTTCTCCACA GAGATCCCTG CCGACTACCA GCGGATATGC 540AAGATGCTCT ACTATCTGTG GATGTTGCAT TCAGTGACTC TGTTTCTGAA CCTGCTTGCC 600TGCCTGGCCT GGTTCTCGGG CAACAGCTCC AAGGGAGTGG ACTTTGGCCT CTCCATCCTG 660TGGTTTCTGA TCTTCACTCC CTGTGCCTTC CTTTGTTGGT ACCGACCCAT CTATAAGGCC 720TTTAGGTCCG ACAACTCTTT CAGCTTCTTT GTGTTCTTCT TTGTATTTTT TTGTCAAATA 780GGGATCTACA TCATCCAGTT GGTTGGCATC CCTGGCCTGG GGGACAGCGG TTGGATTGCA 840GCCCTGTCTA CACTGGATAA TCATTCCCTG GCCATATCAG TCATCATGAT GGTGGTGGCT 900GGCTTCTTCA CCCTCTGTGC CGTGCTCTCA GTCTTCCTCC TGCAGCGGGT GCACTCCCTC 960TACCGACGGA CAGGGGCCAG CTTCCAGCAG GCCCAGGAGG AGTTTTCCCA GGGCATCTTC 1020AGCAGCAGAA CCTTCCACAG AGCTGCTTCA TCTGCTGCCC AAGGAGCCTT CCAGGGGAAT 1080TAGTCCTCCT CTCTTCTCTC CCCCTCAGCC TTTCTCTCGC CTGCCTTCTG AGCTGCACTT 1140TCCGTGGGTG CCTTATGTGG TGGTGGTTGT GCCCAGCACA GACCTGGCAG GGTTCTTGCC 1200GTGGCTCTTC CTCCTCCCTC AGCGACCAGC TCTCCCTGGA ACGGGAGGGA CAGGGAATTT 1260TTTCCCCCTC TATGTACAAA AAAAAACAAA GCTCTCTTTC CTTCTCTGGT GATGGTTTGG 1320TAGGATTCTT TTGTCTCTGG AAGCAGTGGG ACTGAAGTTC TCTTCGTCCT GTGCACACAC 1380AGACACCCCC ACACAGTTGG GATCACAGGC TGACCTGGGC CCATCCCAGC TGGAGCTTTC 1440TGCCAGGGTC CTGGGCCTTG ACTCCCCCAC CCTGCAGGCC TGGCCTGAAT CTGGCTTCTT 1500AGACACAGCC CAGTCCTTCC TGCCTGGGCT GGGAATAAGC CTCTCACAGG TTCTGGTGGA 1560CAGATCTGTT CCCCAGGTCA CTCCAGTGGT CTCCAGGCTT CCAGAGAAGG CTGGTTGCCT 1620CAAGCTCTTC TCTGCCTCAT AAACGGATCC AGAGAAGGCT GGTTGCCTTA AGCTCTTCCC 1680TGCCTCGTGT TCCTGAGAAA CGGATTAATA GCCCTTTATC CCCCTGCACC CTCCTGCAGG 1740GGATGGCACT TTGAGCCCTC TGGAGCCCTC CCCTTGCTGA GCCTTACTCT CTTCAGACTT 1800TCTGAATGTA CAGTGCCGTT GGTTGGGATT TGGGGACTGG AAGGGACCAA GGACACTGAC 1860CCCAAGCTGT CCTGCCTAGC GTCCAGCGTC TTCTAGGAGG GTGGGGTCTG CCTGTCCTGG 1920TGTGGTTGGT TTGGCCCTGT TTGCTGTGAC TACCCCCCCC CCTCCCCGAA CCGAGGGACG 1980GCTGCCTTTG TCTCTGCCTC AGATGCCACC TGCCCCGCCC ATGCTCCCCA TCAGCAGCAT 2040CCAGACTTTC AGGAAGGGCA GGACCAGCCA GTCCAGAACC GCATCCCTCA GCAGGGACTG 2100ATAAGCCATC TCTCGGAGGG CCCCCTAATA CCCAGTGGAG TCTGGTTCAC ACCCTGGGGG 2160GTGTGTCACT GTGATGGGAC ACGTAGGAGT CCACCCTTAA AACCAGCACC CTGTCCCTCG 2220AGGCTGCCGA GTGGGTGTGT GGACTGGGGT GCCTTCCCAC AAAACTAGCC TCCGGCTCTG 2280GGCCCGAGAC AGCCGCAGGC CCCAGCCACT GAATGATACT GGCAGCGGCT GGGGTTTTAT 2340GAACTCCTTT CTGGTATTTT TTCCCCTCTA TGTACAAATG TATATGTTAC GTCTCAATTT 2400TTGTGCTTAA GTAAAAATAA AAACATTTTC AGAC 2434 338 amino acids amino acidsingle linear unknown GenBank 487057 5 Met Ser Asp Phe Asp Ser Asn ProPhe Ala Asp Pro Asp Leu Asn Asn 1 5 10 15 Pro Phe Lys Asp Pro Ser ValThr Gln Val Thr Arg Asn Val Pro Pro 20 25 30 Gly Leu Asp Glu Tyr Asn ProPhe Ser Asp Ser Arg Thr Pro Pro Pro 35 40 45 Gly Gly Val Lys Met Pro AsnVal Pro Asn Thr Gln Pro Ala Ile Met 50 55 60 Lys Pro Thr Glu Glu His ProAla Tyr Thr Gln Ile Thr Lys Glu His 65 70 75 80 Ala Leu Ala Gln Ala GluLeu Leu Lys Arg Gln Glu Glu Leu Glu Arg 85 90 95 Lys Ala Ala Glu Leu AspArg Arg Glu Arg Glu Met Gln Asn Leu Ser 100 105 110 Gln His Gly Arg LysAsn Asn Trp Pro Pro Leu Pro Ser Asn Phe Pro 115 120 125 Val Gly Pro CysPhe Tyr Gln Asp Phe Ser Val Asp Ile Pro Val Glu 130 135 140 Phe Gln LysThr Val Lys Leu Met Tyr Tyr Leu Trp Met Phe His Ala 145 150 155 160 ValThr Leu Phe Leu Asn Ile Phe Gly Cys Leu Ala Trp Phe Cys Val 165 170 175Asp Ser Ser Arg Ala Val Asp Phe Gly Leu Ser Ile Leu Trp Phe Leu 180 185190 Leu Phe Thr Pro Cys Ser Phe Val Cys Trp Tyr Arg Pro Leu Tyr Gly 195200 205 Ala Phe Arg Ser Asp Ser Ser Phe Arg Phe Phe Val Phe Phe Phe Val210 215 220 Tyr Ile Cys Gln Phe Ala Val His Val Leu Gln Ala Ala Gly PheHis 225 230 235 240 Asn Trp Gly Asn Cys Gly Trp Ile Ser Ser Leu Thr GlyLeu Asn Lys 245 250 255 Asn Ile Pro Val Gly Ile Met Met Ile Ile Ile AlaAla Leu Phe Thr 260 265 270 Ala Ser Ala Val Ile Ser Leu Val Met Phe LysLys Val His Gly Leu 275 280 285 Tyr Arg Thr Thr Gly Ala Ser Phe Glu LysAla Gln Gln Glu Phe Ala 290 295 300 Thr Gly Val Met Ser Asn Lys Thr ValGln Thr Ala Ala Ala Asn Ala 305 310 315 320 Ala Ser Thr Ala Ala Thr SerAla Ala Gln Asn Ala Phe Lys Gly Asn 325 330 335 Gln Met

What is claimed is:
 1. A purified polypeptide comprising an amino acidsequence of SEQ ID NO:1 or SEQ ID NO:3.
 2. A composition comprising aneffective amount of a de of claim 1 and a pharmaceutically acceptableexcipient.